This application is the national phase under 35 U.S.C. xc2xa7371 of prior PCT International Application No. PCT/JP97/03579 which has an International filing date of Oct. 7, 1997 which designated the United States of America, the entire contents of which are hereby incorporated by reference.
The present invention relates to a method for detecting nonsense mutations and frameshift mutations.
If a structural gene has a nonsense mutation or a frameshift mutation therein, normal protein is not produced. That is, in case of a nonsense mutation, the amino acid sequence encoded by the region downstream of the mutated site is not produced at all, so that a protein shorter than the normal protein is produced. In case of a frameshift mutation, the amino acid sequence encoded by the region downstream of the mutated site is completely different from that of the normal amino acid sequence. Therefore, it is thought that, in general, existence of a nonsense mutation or a frameshift mutation in a structural gene results in a disease. Thus, it is clinically important to detect nonsense mutations and frameshift mutations in structural genes.
A method for detecting nonsense mutations or frameshift mutations in structural genes is the method for measuring the activities of the proteins encoded by the structural genes. However, measurement of protein activities often requires complicated operations and there are a number of normal proteins which do not have measurable activities. Nonsense mutations and frameshift mutations can also be detected by sequencing the entire test gene. However, this method is complicated and laborious especially when the size of the test gene is large. Mutations of DNAs can also be sensitively detected by PCR-SSCP (single-stranded conformation polymorphism). However, this method necessitates electrophoresis and it is impossible to distinguish nonsense mutations from other point mutations. Further, this method cannot be applied to large DNAs.
Accordingly, an object of the present invention is to provide a method for detecting nonsense mutations and frameshift mutations, which is simple, and which may be applied to large DNAs.
The present inventors intensively studied to discover that nonsense mutations and frameshift mutations may be detected by using as a reporter gene a structural gene encoding a polypeptide detectable based on a function thereof, inserting a test nucleic acid fragment into a site upstream of the reporter gene, which test nucleic acid fragment does not shift the open reading frame of the reporter gene when the test nucleic acid fragment is normal type, expressing the test nucleic acid fragment and the reporter gene downstream thereof, and by determining whether or not a fusion polypeptide having the function of the polypeptide encoded by the reporter gene is produced, thereby completing the present invention.
That is, the present invention provides a method for detecting nonsense mutations and frameshift mutations comprising the steps of inserting a test nucleic acid fragment into a site of a vector having a promoter, a translational initiation codon downstream of the promoter, a reporter gene which is a structural gene located downstream of the translational initiation codon, which is operably linked to the promoter, which encodes a polypeptide, a fusion polypeptide formed by ligating the N-terminal of the polypeptide to another polypeptide being detectable based on a function of the polypeptide encoded by the reporter gene, the site into which the test nucleic acid fragment is inserted being located downstream of the translational initiation codon and upstream of the reporter gene, the test nucleic acid fragment being one which allows, when inserted, in-frame location of the reporter gene with respect to the translational initiation codon when the test nucleic acid is normal type; expressing the test nucleic acid fragment and the reporter gene downstream thereof in the resulting recombinant vector in a host cell, and determining whether the fusion polypeptide having the function of the polypeptide encoded by the reporter gene is produced or not.
By the present invention, a method by which nonsense mutations and frameshift mutations alone may be specifically detected by simple operations was provided. By the present invention, since detection may be made without operations such as electrophoresis, the operations are very simple. Further, by using a eukaryotic cell such as yeast cell as a host, large test nucleic acid fragment up to about 3.5 kb may be examined, so that the number of fragments when a gene is divided may be reduced. Further, since mutations close to PCR-primer franking sequences may be detected, the sizes of the overlapping regions between the divided fragments may be reduced. Still further, mutations of heterozygous genes may also be detected.